WO2018127494A1

WO2018127494A1 - Methods of producing lipolanthipeptides

Info

Publication number: WO2018127494A1
Application number: PCT/EP2018/050076
Authority: WO
Inventors: Dominique Le Beller
Original assignee: Deinobiotics
Priority date: 2017-01-03
Filing date: 2018-01-02
Publication date: 2018-07-12

Abstract

The present invention relates to novel bicyclic antimicrobial lanthipeptides, their corresponding peptide leader sequences, and the production thereof.

Description

METHODS OF PRODUCING LIPOLANTHIPEPTIDES

FIELD OF THE INVENTION

The present invention relates to novel compounds, their production, and the uses thereof. BACKGROUND OF THE INVENTION

The evolution and spread of antibiotic resistance among bacteria is a major public health problem today, especially in the hospital setting with the emergence of multidrug resistant strains. Intensive research efforts have led to the development of new antibiotics effective against these resistant strains. Nevertheless, through use, mechanisms of resistance to these drugs emerge and limit their efficacy.

Infections caused by multidrug-resistant Gram-positive bacteria represent a major public health burden, not just in terms of morbidity and mortality, but also in terms of increased expenditure on patient management and implementation of infection control measures.

In particular, Staphylococcus aureus is one of the most commonly identified pathogens in human medicine and is a major cause of nosocomial infections and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA) was reported for the first time in 1961 and is now widespread in hospitals all over the world.

The increasing burden of Gram-positive infections is not limited to microorganisms within the genus Staphylococcus, but also involves for example Enterococcus spp., in particular with the emergence of vancomycin-resistant enterococci (VRE) strains or Streptococcus spp. with reduced susceptibility to penicillins and macrolides.

Therefore, the search for new chemical entities with antimicrobial properties and structures differing from those found in conventional antibiotics is viewed as a pressing need to develop new ways to curb these resistant infections. SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a bicyclic peptide of formula (I)

O

wherein

Xi, X₂, X3, X₄ and X5 are independently selected and each represents an amino acid, and

B is selected from the group consisting of hydrogen, a peptide chain of a size comprised between 1 and 30 amino acid residues, and an a-amino-protecting group.

B may be hydrogen.

B may also be a peptide chain comprising, or consisting of, the sequence L-(X)io- E (SEQ ID NO: 24) wherein X represents an amino acid independently selected at each occurrence. In particular, B may be a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MSLEQLEALDASSEAAEMA (SEQ ID

NO: 1) MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2),

MID VTNI ADLHDID ATS GA AEL V (SEQ ID NO: 3),

MIDVTDINSLQAIESHSATSELL (SEQ ID NO: 12),

MDIADVMDLQGEEVVADGVELP (SEQ ID NO: 13),

MDLTNVMELQGTEIVADGVELP (SEQ ID NO: 14),

MDLANVMDLQGTEIVADGIELP (SEQ ID NO: 15),

MDLTNVIDLQGTEIVADGVELP (SEQ ID NO: 16) and

MTLEQLEALDASSEAAEMA (SEQ ID NO: 17). More particularly, B may be a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MS LEQLE ALD AS SE A AEM A (SEQ ID NO: 1) MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2) and MID VTNIADLHDID ATS GA AELV (SEQ ID NO: 3).

Preferably,

a) Xi is an amino acid selected from the group consisting of A, G, Q, L,W, S and

T, preferably A or G, more preferably A; and/or

b) X2 is an amino acid selected from the group consisting of R, L, V, I, G, T, A, and S, preferably from L, V, I, G and A, even more preferably from L, V, I and A, more preferably L or I, and even more preferably I; and/or

c) X3 is an amino acid selected from the group consisting of G, S, A, C, L, V, T,

P and I, preferably from G, S, A and T, more preferably G or S, and more preferably S; and/or

d) X₄ is an amino acid selected from the group consisting of I, Q, S, N, E, D, W, H, P and T, preferably Q or N, more preferably N; and/or

e) X5 is an amino acid selected from the group consisting of G, A, S, T, N, R, H,

P and D, preferably from G, A, S and T, more preferably G or S, even more preferably G.

More preferably,

Xi is an amino acid selected from the group consisting of A and G, preferably is A, and/or

X2 is an amino acid selected from the group consisting of L, V, I, G, A, R, T and S, preferably from the group consisting of V, T, A and G, and/or

X3 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of A and S, and/or

X₄ is an amino acid selected from the group consisting of Q, N, I, S, E, D, W, H,

P and T, preferably from the group consisting of S, E and D, and/or

X5 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G and T.

Alternatively,

Xi is an amino acid selected from the group consisting of A and G, preferably is

A, and/or

X2 is an amino acid selected from the group consisting of L, V, I, G, A, R, T and S, preferably from the group consisting of L, V, I, G, A and T, more preferably from the group consisting of L, V, I, G and A, and even more preferably from the group consisting of L or I, and/or

X3 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G, A and S, and more preferably from the group consisting of G and S, and/or

X4 is an amino acid selected from the group consisting of Q, N, I, S, E, D, W, H, P and T, preferably from the group consisting of Q, N, S, E and D, more preferably from the group consisting of Q and N, and/or

X5 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G, S and T, more preferably from the group consisting of G and S.

Alternatively,

A,

X2 is an amino acid selected from the group consisting of V, I, G, T and A,

X3 is an amino acid selected from the group consisting of A and S,

X4 is an amino acid selected from the group consisting of N, S, E and D, and

X5 is an amino acid selected from the group consisting of G and T.

In particular, Xi may be A, X2 may be L and, X3 may be G.

Alternatively, Xi may be A, X3 may be G and X4 may be Q.

Preferably, Xi is A, X2 is L, X3 is G, X₄ is Q and X5 is S, or

Xi is A, X2 is I, X3 is S, X₄ is N and X5 is G, or

Xi is A, X2 is V, X3 is S, X₄ is S and X5 is G, or

Xi is A, X2 is T, X3 is A, X₄ is D and X5 is G, or

Xi is A, X₂ is T, X₃ is S, X₄ is D and X₅ is G, or

Xi is A, X2 is A, X3 is S, X₄ is E and X5 is T, or

Xi is A, X₂ is G, X₃ is S, X₄ is E and X₅ is G.

In another aspect, the present invention also relates to a method of producing a bicyclic peptide of the invention, comprising:

a) providing a compound of formula (V)

wherein Xi, X₂, X₃, X₄ and X₅ are as defined above, and Z is a lipophilic moiety, and

b) cleaving Z, thereby producing a bicyclic peptide of formula (I),

and optionally recovering the bicyclic peptide.

The compound in step a) may be obtained from a microorganism producing said compound. The microorganism may naturally produce said compound or may be genetically modified to produce said compound.

Preferably, step b) is enzymatically performed, more preferably using a deacylase, and even more preferably an aculeacin-A deacylase produced by Actinoplanes utahensis NRRL 12052.

In a further aspect, the present invention relates to a method of producing a bicyclic peptide of the invention, comprising:

providing a linear peptide comprising, or consisting of, the sequence B-X1-S-X2- X3-S-X4-X5-C (SEQ ID NO: 4), wherein B is a peptide chain of a size comprised between 1 and 30 amino acid residues, and Xi, X₂, X₃, X₄ and X5 are as defined above, and

contacting said linear peptide with an enzyme or enzymatic extract derived from a microorganism producing a bicyclic peptide.

In another aspect, the present invention relates to a peptide comprising, or consisting of, the sequence B-X1-S-X2-X3-S-X4-X5-C (SEQ ID NO: 4), wherein B is a hydrogen, a peptide chain of a size of between 1 and 30 amino acid residues, or an a- amino-protecting group; and Xi, X₂, X₃, X₄ and X5 are independently selected and each represents an amino acid. In another aspect, the present invention relates to a method of producing a lipolanthipeptide, preferably an antimicrobial lipolanthipeptide, comprising:

providing a bicyclic peptide of the invention, and

adding a lipophilic moiety to said peptide.

The lipophilic moiety may be linked to B, optionally in replacement of B.

In particular, the lipophilic moiety may be R4-W-L- wherein

L is a bifunctional linker, preferably selected from the group consisting of -C(=0)- , -S02-, -CS-, -0-CS-, -NH-CS-, -PO-, -OPO-, -OC(=0)- and -NHCO-,

W is a C6-C20 saturated or unsaturated linear hydrocarbon chain, said chain being optionally (i) interrupted by one or several heteroatoms independently selected from N, S and O, and/or (ii) interrupted by one or several groups independently selected from a phenyl group and a 5 or 6-membered-ring heterocycle, said phenyl group or heterocycle being optionally substituted by one or several groups independently selected from C1-C3 alkyl groups, -OH and C1-C3 alkoxy groups, and/or (iii) substituted by one or several groups independently selected from C1-C3 alkyl groups, halogens, -OH, methoxy or acetoxy, and

P4 is selected from the group consisting of hydrogen and a basic group.

In a last aspect, the present invention also relates to a microorganism genetically modified to express a heterologous gene encoding a linear peptide comprising, or consisting of, the sequence B-X1-S-X2-X3-S-X4-X5-C (SEQ ID NO: 4), wherein Xi, X₂, X3, X₄ and X5 are independently selected and each represents an amino acid, preferably as defined above, and B is selected from the group consisting of hydrogen and a peptide chain of a size comprised between 1 and 30 amino acid residues. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: LC-MS spectra- Crude extract from Nocardia altamirensis .

Figure 2: MS-Spectra - Crude extract from Nocardia altamirensis.

Figure 3: LC-MS analysis of the fraction containing compound NOC1 from Nocardia terpenica.

Figure 4: MS/MS spectra of compound NOC1 from Nocardia terpenica. Figure 5 : LC-UV analysis of the reaction mixture after 22 hours. The core peptide corresponds to the peak of RT : 1.18 min. Compound A corresponds to the peak of RT : 6.98 min.

Figure 6: LC-MS analysis of the reaction mixture after 22 hours. The core peptide corresponds to the peak of RT : 1.12 min. Compound A corresponds to the peak of RT : 7.05 min.

Figure 7: MS spectra of the core peptide (RT : 1.12 min).

Figure 8: MS/MS spectra of the core peptide (RT : 1.12 min).

Figure 9: MS spectra of the reacylated core peptide.

Figure 10: ¾ NMR spectra of compound Noel in CD₃CN:D₂0 60:40.

Figure 11: COSY ^lH -^lH NMR spectra of compound Noel in CD₃CN:D₂0 60:40. Figure 12: HSQCY ¾ -¹³C RMN spectra of compound Noel in CD₃CN:D₂0

60:40.

Figure 13: Intra-residual fatty acid chain NMR assignment of compound Noel. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates novel compounds, their production, as well as the uses thereof. The invention also relates to methods for producing antimicrobial molecules using said compounds.

The inventors have designed a novel class of antimicrobial agents. These agents typically comprise a core polycyclic peptide and a fatty acid moiety. Based on their structure, these new compounds are herein referred to as lipolanthipeptide. Such compounds exhibit potent antimicrobial activity, particularly against Gram positive bacteria, including vancomycin-resistant Enterococcus strains or methicillin-resistant Staphylococcus strains, as well as against mycobacteria and pathogenic fungi such as Candida strains.

The present invention relates to methods of producing such lipolanthipep tides, and in particular to hemi-synthetic methods for producing such compounds. These methods involve obtaining a core peptide structure, either from biological sources, e.g. from microorganisms, and/or through enzymatic and/or chemical synthesis, and adding a lipophilic moiety to said core peptide structure to produce lipolanthipep tides. The invention also relates to intermediate compounds and reagents suitable for use in such method, as well as to their manufacture.

Accordingly, in a first aspect, the present invention relates to a bicyclic peptide of formul

wherein

As used herein, the term "amino acid" or "amino acid residue" refers to any of the naturally occurring amino acids, including rare amino acids, as well as non-natural amino acid analogues.

In preferred embodiments, the term "amino acid" refers to any of the 20 naturally occurring amino acids which may be represented by their one-letter code according to the following nomenclature: A: alanine, C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan and Y: tyrosine. In some embodiments, the side chains of these amino acid residues may be chemically modified, for example by glycosylation, amidation, acylation, acetylation or methylation.

The amino acids may be in the L or D configuration, or a combination of both. In preferred embodiments, Xi, X₂, X₃, X₄ and X5 represent amino acids in the L configuration.

The amino acid residues may be linked to the adjacent components through "classical" CONH peptide bonds or through pseudo-peptide bonds. In particular, the compound of the invention may comprise one or several pseudo-peptide bonds replacing one or several CONH peptide bonds.

In preferred embodiments, Xi, X₂, X₃, X₄ and X5 are linked to the adjacent components through "classical" CONH peptide bonds and the compound of the invention is of formula (II)

wherein

B has the same meaning as described above and

SCi, SC2, SC₃, SC₄ and SC5 represent the side-chains of the amino acids Xi, X₂, X₃, X₄ and X5, respectively.

The compound of formula (I) or (II) has preferably one or several of the following features:

a) Xi is an amino acid selected from the group consisting of A, G, Q, L,W, S and T, preferably A or G, more preferably A; and/or b) X2 is an amino acid selected from the group consisting of R, L, V, I, G, T, A, and S, preferably from L, V, I, G and A, even more preferably from L, V, I and A, more preferably L or I, and even more preferably I; and/or

c) X3 is an amino acid selected from the group consisting of G, S, A, C, L, V, T, P and I, preferably from G, S, A and T, more preferably G or S, and more preferably S; and/or

e) X5 is an amino acid selected from the group consisting of G, A, S, T, N, R, H, P and D, preferably from G, A, S and T, more preferably G or S, even more preferably

G.

More preferably, the compound of formula (I) or (II) has preferably one or several of the following features:

a) Xi is an amino acid selected from the group consisting of A and G, preferably is A, and/or

b) X2 is an amino acid selected from the group consisting of L, V, I, G, A, R, T and S, preferably from the group consisting of L, V, I, G, A and T, more preferably from the group consisting of L, V, I, G and A, and even more preferably from the group consisting of L or I and/or

c) X3 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G, A and S, and more preferably from the group consisting of G and S, and/or

d) X₄ is an amino acid selected from the group consisting of Q, N, I, S, E, D, W, H, P and T, preferably from the group consisting of Q, N, S, E and D, more preferably from the group consisting of Q and N, and/or

e) X5 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G, S and T, more preferably from the group consisting of G and S.

Alternatively, the compound of formula (I) or (II) has one or several of the following features: Xi is an amino acid selected from the group consisting of A and G, preferably is A, and/or

X₄ is an amino acid selected from the group consisting of Q, N, I, S, E, D, W, H, P and T, preferably from the group consisting of S, E and D, and/or

In a particular embodiment, the compound of formula (I) or (II) has one or several of the following features:

X2 is an amino acid selected from the group consisting V, T, A and G, and/or X3 is an amino acid selected from the group consisting of A and S, and/or X4 is an amino acid selected from the group consisting of S, E and D, and/or X5 is an amino acid selected from the group consisting of G and T.

Alternatively, the compound of formula (I) or (II) has preferably one or several of the following features:

a) Xi is an amino acid selected from the group consisting of A or G, preferably is A; and/or

b) X2 is an amino acid selected from the group consisting of L, V, I, G and A, preferably an amino acid selected from the group consisting of L, V, I and A, more preferably is selected from the group consisting of L and I, and even more preferably is I; and/or

c) X3 is an amino acid selected from the group consisting of G, A, S and T, preferably an amino acid selected from the group consisting of G and S, and more preferably is S; and/or

d) X₄ is an amino acid selected from the group consisting of Q and N, preferably is N; and/or e) X5 is an amino acid selected from the group consisting of G, A, S and T, preferably is an amino acid selected from the group consisting of G and S, and more preferably is G. The compound of formula (I) or (II) may meet one feature, two features [for instance a) and b); a) and c); a) and d); a) and e); b) and c); b) and d); b) and e); c) and d); c) and e); d) and e)], three features [for instance a), b) and c); a), b) and d); a), b) and e); a), c) and d); a), c) and e); a), d) and e); b), c) and d); b), c) and e); c), d) and e)], four features [a), b), c) and d); a), b), c) and e); a), b), d) and e); a), c), d) and e); b), c), d) and e)], or five features [i.e. a), b), c), d) and e)] as described above.

In a particular embodiment,

X2 is an amino acid selected from the group consisting of L, V, I, G, T and A, X3 is an amino acid selected from the group consisting of G, A and S,

X4 is an amino acid selected from the group consisting of Q, N, S, E and D, and X5 is an amino acid selected from the group consisting of G, S and T.

In a particular embodiment,

X2 is an amino acid selected from the group consisting of V, G, T and A, X3 is an amino acid selected from the group consisting of A and S,

X₄ is an amino acid selected from the group consisting of S, E and D, and X5 is an amino acid selected from the group consisting of G and T.

In another particular embodiment,

Xi is an amino acid selected from the group consisting of A and G, preferably is A,

X3 is an amino acid selected from the group consisting of S and G, and

X5 is an amino acid selected from the group consisting of S and G. Optionally, X2 is an amino acid selected from the group consisting of L, V, I, G, T and A, and/or X₄ is an amino acid selected from the group consisting of Q, N, S, E and D. In another particular embodiment,

Xi is an amino acid selected from the group consisting of A and G,

X2 is an amino acid selected from the group consisting of L, V, I, G and A, X3 is an amino acid selected from the group consisting of G, A, S and T, X4 is an amino acid selected from the group consisting of Q, I and N, and X5 is an amino acid selected from the group consisting of G, A, S and T.

In another particular embodiment,

A,

X2 is an amino acid selected from the group consisting of L, V and I, preferably selected from the group consisting of L and I,

X3 is an amino acid selected from the group consisting of G and S,

X4 is an amino acid selected from the group consisting of Q, I and N, preferably is Q or N, and

X5 is an amino acid selected from the group consisting of G and S, preferably is

G.

In another particular embodiment,

X2 is an amino acid selected from the group consisting of L, V, I, G and A, X3 is an amino acid selected from the group consisting of G, A, S and T, X4 is an amino acid selected from the group consisting of Q and N, and

X5 is an amino acid selected from the group consisting of G, A, S and T.

In a more particular embodiment,

A, X2 is an amino acid selected from the group consisting of L, V and I, preferably selected from the group consisting of L and I, more preferably is I,

X3 is an amino acid selected from the group consisting of G and S, preferably is

S,

X₄ is an amino acid selected from the group consisting of Q and N, preferably is

N, and

G.

In another particular embodiment,

Xi is A,

X2 is an amino acid selected from the group consisting of G, T and A, preferably G and T,

X3 is an amino acid selected from the group consisting of S and A, preferably is

A,

X4 is an amino acid selected from the group consisting of D and E, and

X5 is an amino acid selected from the group consisting of T and G, preferably is

T.

In another particular embodiment,

A,

X2 is an amino acid selected from the group consisting of V, I, G, T and A, X3 is an amino acid selected from the group consisting of A and S,

X₄ is an amino acid selected from the group consisting of N, S, E and D, and X5 is an amino acid selected from the group consisting of G and T.

In a further particular embodiment, X2 is L and X3 is G.

In another particular embodiment, X3 is G and X₄ is I or Q.

In another particular embodiment, X3 is G and X₄ is Q.

In a further particular embodiment, X₄ is Q and X5 is S.

In another particular embodiment, X2 is L, X3 is G and X₄ is I.

In another particular embodiment, X2 is L, X3 is G and X₄ is Q.

In another particular embodiment, Xi is A, X2 is L and, X3 is G. In another particular embodiment, Xi is A, X3 is G and X₄ is Q.

In another particular embodiment, Xi is A, X2 is T, X₄ is D and X5 is G.

In another particular embodiment, Xi is A and X3 is S.

In another particular embodiment, Xi is A and X5 is G.

In another particular embodiment, Xi is A, X3 is S and X5 is G. Optionally, in this embodiment, X2 is an amino acid selected from the group consisting of L, V, I, G, T and A, and/or X₄ is an amino acid selected from the group consisting of Q, N, S, E and D.

In a preferred embodiment, Xi is A, X2 is L, X3 is G, X₄ is Q and X5 is S.

mpound is of formula (III)

(III)

wherein B is as defined above and hereafter.

In another preferred embodiment, Xi is A, X2 is I, X3 is S, X₄ is N and X5 is G. Preferably, in this embodiment, the compound is of formula (IV)

wherein B is as defined above and hereafter.

In another preferred embodiment, Xi is A, X2 is V, X3 is S, X₄ is S and X5 is G. In another preferred embodiment, Xi is A, X2 is T, X3 is A, X₄ is D and X5 is G.

In another preferred embodiment, Xi is A, X2 is T, X3 is S, X₄ is D and X5 is G. In another preferred embodiment, Xi is A, X2 is A, X3 is S, X₄ is E and X5 is T. In another preferred embodiment, Xi is A, X2 is G, X3 is S, X₄ is E and X5 is G. Preferably, in all embodiments described herein, Xi is A.

In an embodiment, B is hydrogen.

In another embodiment, B is an a-amino-protecting group. Such protecting group is particularly interesting where the core peptide is subjected to further chemical modification(s) that could affect the a-amino functionality, α-amino-protecting groups, routinely used in peptide chemistry and synthesis may be selected for example from 9- fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), Nps (2- nitrophenylsulfenyl) and Bpoc [2-(4-biphenyl)isopropoxycarbonyl] groups.

In embodiments wherein B is an α-amino-protecting group, said group may be easily removed when needed using any method well known by the skilled person and routinely used in chemical synthesis.

In another embodiment, B is a peptide chain of a size comprising from 1 to 30 amino acid residues, preferably a size comprised between 10 and 30 amino acid residues, more preferably between 15 and 30 amino acid residues, and even more preferably between 15 and 25 amino acid residues. In some preferred embodiments, B is a peptide chain of a size comprising from 19 to 23 amino acid residues. This amino acid chain, when present, may be or contain a tag suitable for purification, or a leader sequence.

In this regard, the natural biosynthesis of lipolanthipeptide antibiotics is initiated with a ribosomally generated linear precursor peptide encoded by a structural gene. This precursor peptide contains an N-terminal leader peptide fused to a core peptide which is then cyclized. The peptides of the invention may thus contain such a leader sequence, or variants thereof.

The sequence of a precursor of a lipolanthipeptide produced by Microbacterium arborescens CIP 55.8 IT (Collection Institut Pasteur) (cf. example 2), is MSLEOLEALDASSEAAEMAASLGSOSC (SEQ ID NO: 5), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MSLEQLEALDASSEAAEMA (SEQ ID NO: 1).

The sequence of the precursor of the lipolanthipeptide NOC1 produced by

Nocardia terpenica (cf. example 1) is (MIDVTNIAELHELDSTSASAELVASISSNGC; SEQ ID NO: 6), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MID VTNI AELHELD STS AS AELV (SEQ ID NO: 2).

The sequence of the precursor of the lipolanthipeptides NOC2 to NOC10 produced by Nocardia altamirensis (cf. example 1) is (MIDVTNIADLHDIDATSGAAELVASISSNGC; SEQ ID NO: 7), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MID VTNIADLHDID ATS GAAELV (SEQ ID NO: 3).

The sequence of the precursor of the antimicrobial compound(s) of formula (I) produced by Tsukamurella sp. 1534 is (MID VTDINSLQ AIESHS ATSELLAS VSS SGC ; SEQ ID NO: 18), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MID VTDINSLQ AIESHSATSELL (SEQ ID NO: 12).

The sequence of the precursor of the antimicrobial compound(s) of formula (I) produced by Streptomyces aureus or Streptomyces flavochromogenes is (MDLTNVIDLQGTEIVADGVELPASGSSEGC; SEQ ID NO: 22), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MDLTNVIDLQGTEIVADGVELP (SEQ ID NO: 16).

The sequence of the precursor of the antimicrobial compound(s) of formula (I) produced by Streptomyces natalensis is (MDLTNVMELQGTEIVADGVELPASTSSDGC: SEQ ID NO: 20), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MDLTNVMELQGTEIVADGVELP (SEQ ID NO: 14).

The sequence of the precursor of the antimicrobial compound(s) of formula (I) produced by Nocardiopsis chromatogenes is (MDIADVMDLQGEEVVADGVELPASTASDGC: SEQ ID NO: 19), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MDIADVMDLQGEEVVADGVELP (SEQ ID NO: 13).

The sequence of the precursor of the antimicrobial compound(s) of formula (I) produced by Nonomuraea Candida is (MDLANVMDLQGTEIVADGIELPASASSETC: SEQ ID NO: 21), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MDLANVMDLQGTEIVADGIELP (SEQ ID NO: 15).

The sequence of the precursor of the antimicrobial compound(s) of formula (I) produced by Microbacterium arborescens strain ND21 is (MTLEOLEALDASSEAAEMAASLGSOSC: SEQ ID NO: 23), wherein the core peptide is underlined. Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence MTLEQLEALDASSEAAEMA (SEQ ID NO: 17).

In an embodiment, B is a peptide chain comprising, or consisting of, the sequence L-(X)io-E (SEQ ID NO: 24) wherein X represents an amino acid independently selected at each occurrence. Preferably, B is a peptide chain comprising, or consisting of, the sequence (X)_n-L-(X)io-E-(X)₂ (SEQ ID NO: 25), wherein n represents an integer selected from 4 to 10, preferably from 5 to 9. In particular, n may be an integer selected from 5, 8 and 9.

In a particular embodiment, B is a peptide chain comprising, or consisting of, the sequence [L/I/V]-(X)2-L-(X)₃-[D/E]-(X)2-[S/A]-(X)₃-E-[L/M] (SEQ ID NO: 26), preferably the sequence [L/I/V]-X-[E/D/S/Q]-L-[H/Q/E]-(X)₂-[D/E]-(X)₂-[S/A]-(X)₃-E- [L/M] (SEQ ID NO: 27). In another particular embodiment, B is a peptide chain comprising, or consisting of, the sequence D-(X)₆-L-(X)io-E-L (SEQ ID NO: 28), preferably the sequence [M/I]- D-[V/L]-[A/T]-[N/D]-[I/V]-(X)₂-L-[H/Q]- (X)₂-[D E]- (X)₂-[S/A]- (X)₃-E-L (SEQ ID NO: 29), more preferably the sequence [M/I]-D-[V/L]-[A/T]-[N/D]-[I/V]-X-[E/D/S]-L- [HQ]-(X)₂-[D/E]- (X)₂-[S/A]- (X)₃-E-L (SEQ ID NO: 30).

In another particular embodiment, B is a peptide chain comprising, or consisting of, the sequence M-I-D-V-T-X-I-(X)₂-L-(X)6-S-(X)₃-E-L (SEQ ID NO: 31), preferably the sequence M-I-D-V-T-[D/N] -I-(X)₂-L-[H/Q] -X- [L/I] - [D/E] - [ A/S ] -X-S -X- [T/A/S ] - [A/SJ-E-L (SEQ ID NO: 32).

In a further particular embodiment, B is a peptide chain comprising, or consisting of, the sequence M-D-(X)₃-V-(X)₂-L-Q-G-X-E-X-V-A-D-G-X-E-L-P (SEQ ID NO: 33), preferably the sequence M-D-[L/I]-[T/A]-[N/D]-V-[I/M]-[D/E]-L-Q-G-X-E-[I/V]-V-A- D-G-[V/I]-E-L-P (SEQ ID NO: 34).

In a more particular embodiment, B is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO: 1 to 3 and 12 to 17, preferably selected from SEQ ID NOs: 12 to 17, more preferably from SEQ ID NOs: 12 to 16, or variants thereof. Preferably, said variants have at least 50%, 60%, 70%, 80% or 90% sequence identity to the reference sequence, more preferably have at least 80% or 90% sequence identity to the reference sequence. In particular embodiments, said variants exhibit 1, 2, 3, 4 or 5 amino acid changes, preferably 1, 2 or 3 amino acid changes, to the reference sequence.

In preferred embodiments, said variants have at least 50%, 60%, 70%, 80% or 90% sequence identity to the reference sequence and comprise, or consist of, the sequence L-(X)io-E (SEQ ID NO: 24), and more preferably comprises, or consists of, the sequence (X)_n-L-(X)io-E-(X)₂ (SEQ ID NO: 25), wherein n represents an integer selected from 4 to 10, preferably from 5 to 9.

Thus, in a particular embodiment, B is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO: 1 to 3 and 12 to 17, preferably selected from SEQ ID NOs: 12 to 17, more preferably from SEQ ID NOs: 12 to 16, and variants thereof having at least 50%, preferably at least 70%, 80%, or 90%, identity to any of these sequences and comprising, or consisting of, SEQ ID NO: 24 or 25, wherein n represents an integer selected from 4 to 10, preferably from 5 to 9. In a more particular embodiment, B is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of SEQ ID NO: 1 to 3 and 12 to 17, preferably selected from SEQ ID NOs: 12 to 17, more preferably from SEQ ID NOs: 12 to 16.

In a more particular embodiment, B is or comprises an amino acid sequence selected from SEQ ID NOs: 1 to 3, or variants thereof having at least 90% identity to any of these sequences. More preferably, B is or comprises an amino acid sequence selected from SEQ ID NOs: 1 to 3, or variants thereof having 1 or 2 amino acid changes. In embodiments wherein B is a peptide chain, said chain may be enzymatically or chemically cleaved when needed. In particular, said chain may be cleaved using a protease. Alternatively, the cleavage may be performed using cyanogen bromide. Cyanogen bromide can cleave a peptide after Met residue. Thus, in this embodiment, the sequence B has to comprise a Met residue at its C-terminal extremity. In particular, the sequence B may comprise, or consist of, the sequence (X)_n-Leu-(X)io-Glu-X-Met (SEQ ID NO: 35), wherein X represents an amino acid independently selected at each occurrence, and n represents an integer selected from 4 to 10, preferably from 5 to 9. All embodiments described above for sequence B and applicable to this generic sequence are also encompassed.

The bicyclic peptide of the invention may be obtained by any method known by the skilled person including classical chemical synthesis (in solid phase or homogeneous liquid phase), enzymatic synthesis, recombinant expression, biological sources, or combinations thereof.

Chemical synthesis may generally be performed using standard solution phase or solid phase peptide synthesis techniques, in which a peptide linkage occurs through the direct condensation of the amino group of one amino acid with the carboxy group of the other amino acid with the elimination of a water molecule. Peptide bond synthesis by direct condensation, as formulated above, requires suppression of the reactive character of the amino group of the first and of the carboxyl group of the second amino acid. The masking substituents must permit their ready removal, without inducing breakdown of the labile peptide molecule. The linear peptide obtained by chemical synthesis may be further cyclized through chemical and/or enzymatic reactions. In a further aspect, the present invention also relates to a method of producing the bicyclic peptide of the invention, said method comprising :

a) providing a compound of formula (V)

wherein Xi, X₂, X3, X₄ and X5 are as defined above and Z is a lipophilic moiety, and

b) cleaving Z, thereby producing a bicyclic peptide of formula (I) or (II). step a) is a compound of formula (VI)

o (VI)

wherein

SCi, SC2, SC3, SC₄ and SC5 represent the side-chains of the amino acids Xi, X₂, X3, X₄ and X5, respectively, and Z is as defined above and hereafter. The compound of formula (V) or (VI) may be a natural compound, a natural compound subjected to biological, chemical or enzymatic modifications, or a synthetic compound. In particular, the lipophilic moiety of such compound may be a naturally occurring moiety, a modified naturally occurring moiety or a synthetic one.

In an embodiment, Z is a fatty acid chain with a terminal carbon optionally substituted by a basic group. Preferably, Z is fatty acid chain with a terminal carbon substituted by a basic group.

ly a group Ri which is

with P2 and R3 being independently selected from hydrogen and C1-C3 alkyl groups, preferably selected from hydrogen and methyl, and more preferably being methyl.

In particular,

R2 and R3 may be hydrogen,

R2 may be hydrogen and R3 methyl and vice-versa, or

R2 and R3 may be methyl.

In a preferred embodiment, R2 and R3 are methyl. In a particular embodiment, Z may be represented as follow:

wherein Y is a saturated or unsaturated linear hydrocarbon chain, said chain being optionally interrupted or substituted, and Ri is as defined above.

Preferably, Y is a C6-C20 saturated or unsaturated linear hydrocarbon chain, preferably uninterrupted and unsubstituted chain.

By "C6-C20 saturated linear hydrocarbon chain" is meant a linear hydrocarbon chain having from 6 to 20 carbons, i.e. 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons, and which does not comprise any unsaturation i.e. any double nor triple bonds. By "C6-C20 unsaturated linear hydrocarbon chain" is meant a linear hydrocarbon chain having from 6 to 20 carbons and which comprises at least one unsaturation i.e. at least one double bond and/or at least one triple bond, preferably at least one double bond.

In the case of an unsaturated linear hydrocarbon chain comprising several unsaturations, each unsaturation may be a triple bond or a double bond. Preferably each unsaturation is a double bond. The double bonds may have indifferently trans configuration (E) or cis configuration (Z). Preferably, the double bond(s) is/are in cis configuration.

Preferably, in embodiments wherein Y is an unsaturated hydrocarbon chain, said chain comprises from 1 to 4 double bonds, more preferably from 1 to 3 double bonds, and even more preferably one or two double bonds.

In particular, Y may be an unsaturated linear hydrocarbon chain comprising one, two or three double bonds. Preferably, Y is selected from the group consisting of

(i) -(CH₂)4-(CH2)_m-CH=CH-(CH2)n-CH=CH-(CH2)p-CH=CH-(CH₂)_q-, wherein m, n, p and q are independently selected from 0 and integers from 1 to

10, and 0 < m+n+p+q < 10, preferably from 0 and integers from 1 to 6, and 0 < m+n+p+q < 6, and more preferably from 0 and integers from 1 to 3, and 0 < m+n+p+q < 3;

(ii) -(CH₂)4-(CH₂)_m-CH=CH-(CH₂)n-CH=CH-(CH₂)_p-,

wherein m, n and p are independently selected from 0 and integers from 1 to 12, and 0 < m+n+p < 12, preferably from 0 and integers from 1 to 8, and 0 < m+n+p < 8, and more preferably from 0 and integers from 1 to 5, and 0 < m+n+p < 5;

(iii) -(CH₂)4-(CH₂)_m-CH=CH-(CH₂)n-,

wherein m and n are independently selected from 0 and integers from 1 to 14, and 0 < m+n < 14, preferably from 0 and integers from 1 to 10, and 0 < m+n < 10, more preferably from 0 and integers from 1 to 7, and 0 < m+n < 7.

In an embodiment, Y is a C₆-Ci8 saturated or unsaturated linear hydrocarbon chain, preferably a C15-C18 saturated or unsaturated linear hydrocarbon chain, i.e. a C15, Ci6 , Ci7 or Cis saturated or unsaturated linear hydrocarbon chain, more preferably a C15- Cis saturated linear hydrocarbon chain.

In another embodiment, Y is a C₆-Ci6 saturated or unsaturated linear hydrocarbon chain, preferably a C9-C16 saturated or unsaturated linear hydrocarbon chain. In a particular embodiment, Y is a C14-C16 saturated or unsaturated linear hydrocarbon chain, preferably a C14 or C½ saturated or unsaturated linear hydrocarbon chain.

Preferably, Y is selected from the group consisting of C½ and C14 saturated linear hydrocarbon chains and C16 and C14 unsaturated linear hydrocarbon chains comprising one, two or three double bonds, preferably one double bond.

In particular, the C16 and C14 unsaturated linear hydrocarbon chains comprising one, two or three double bonds may be selected from the group consisting of

(i) -(CH₂)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)p-CH=CH-(CH₂)_q-, wherein m, n, p and q are independently selected from 0 and integers from 1 to 6, and m+n+p+q

= 4 or 6, preferably 2 < q ;

(ii) -(CH₂)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH₂)p-, wherein m, n and p are independently selected from 0 and integers from 1 to 8, and m+n+p = 6 or 8, preferably 2 < p; and

(iii) -(CH₂)4-(CH₂)_m-CH=CH-(CH₂)n-, wherein m and n are independently selected from 0 and integers from 1 to 10, and m+n = 8 or 10, preferably 2 < n.

More preferably, Y is selected from the group consisting of C½ and C14 saturated linear hydrocarbon chains and C½ unsaturated linear hydrocarbon chains comprising one double bond, preferably -(CH₂)4-(CH₂)_m-CH=CH-(CH₂)_n-, wherein m and n are independently selected from 0 and integers from 1 to 10, and m+n = 10, preferably 2 < n.

Preferably, in said embodiment, the compound is of formula (X)

O (X) In another embodiment, Y is a C₆-Ci3 saturated or unsaturated linear hydrocarbon chain, preferably a C9-C 13 saturated or unsaturated linear hydrocarbon chain.

In a particular embodiment, Y is a C10-C12 saturated or unsaturated linear hydrocarbon chain, preferably a C10 or C12 saturated or unsaturated linear hydrocarbon chain.

Preferably, Y is selected from the group consisting of C10 and C12 saturated linear hydrocarbon chains and C10 and C12 unsaturated linear hydrocarbon chains comprising one, two or three double bonds.

In particular the C10 and C12 unsaturated linear hydrocarbon chains comprising one, two or three double bonds may be selected from the group consisting of

(i) -(CH2)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)p-CH=CH-(CH₂)_q-, wherein m, n, p and q are independently selected from 0 and integers from 1 to 2, and m+n+p+q = 0 or 2;

(ii) -(CH2)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH₂)p-, wherein m, n and p are independently selected from 0 and integers from 1 to 4, and m+n+p = 2 or 4; and

(iii) -(CH2)4-(CH2)m-CH=CH-(CH2)n-, wherein m and n are independently selected from 0 and integers from 1 to 6, and m+n = 4 or 6.

More preferably, Y is selected from the group consisting of

(i) a Cio saturated linear hydrocarbon chain,

(ii) a Cio unsaturated linear hydrocarbon chains comprising one or two double bonds, preferably selected from

-(CH2)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH₂)p-, wherein m, n and p are independently selected from 0 and integers from 1 to 2, and m+n+p = 2, and -(CH2)4- (CH2)m-CH=CH-(CH2)n-, wherein m and n are independently selected from 0 and integers from 1 to 4, and m+n = 4; and

(iii) a C12 unsaturated linear hydrocarbon chains comprising three double bonds, preferably -(CH2)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)p-CH=CH-(CH₂)_q-, wherein m, n, p and q are independently selected from 0 and integers from 1 to 2, and m+n+p+q = 2.

Preferably, in said embodiment, the compound is of formula (XI)

In a particular embodiment, Y is selected from the group consisting of Cio, C12, Ci4 and C½ saturated or unsaturated linear hydrocarbon chains.

In a more particular embodiment, Y is selected from the group consisting of Cio,

Ci4 and C½ saturated linear hydrocarbon chains, C½ unsaturated linear hydrocarbon chains comprising one double bond, Cio unsaturated linear hydrocarbon chains comprising one or two double bonds, and C12 unsaturated linear hydrocarbon chains comprising three double bonds. Preferably, in embodiments wherein Y is a C12 unsaturated linear hydrocarbon chain comprising three double bonds, Y is -(CH2)₄- CH=CH-(CH₂)2-CH=CH-CH=CH-.

Preferably in said embodiment, the compound is of formula (X) or (XI).

The compound provided in step a) is preferably obtained by culturing a microorganism producing said compound under conditions suitable to produce said compound, and recovering said compound from the culture.

The microorganism producing the compound may naturally produce said compound or may be genetically modified to produce said compound.

In an embodiment, the microorganism producing the compound is a microorganism naturally producing said compound. Preferably, the microorganism is selected from the group consisting of Nocardia, Microbacterium, Tsukamurella, Streptomyces, Nocardiopsis and Nonomuraea bacteria.

More preferably, the microorganism is selected from the group consisting of Nocardia terpenica, preferably Nocardia terpenica DSMZ 44935, Nocardia altamirensis , preferably Nocardia altamirensis DSMZ 44997, Microbacterium arborescens, preferably Microbacterium arborescens CIP 55.81T (Collection Institut Pasteur) or Microbacterium arborescens strain ND21 (NCBI BioSample: SAMN05211039), Microbacterium sp. TS-1 (Uniprot taxon identifier : 1344956), Tsukamurella sp. 1534 (Oulmi et al. J Bacterid. 2012 Oct; 194(19): 5482-5483), Streptomyces aureus, preferably Streptomyces aureus DSM 41785, Streptomyces flavochromogenes, preferably Streptomyces flavochromogenes DSM 40541, Streptomyces natalensis, preferably Streptomyces natalensis DSM 40357, Nocardiopsis chromatogenes, preferably Nocardiopsis chromatogenes DSM 44844 and Nonomuraea Candida, preferably Nonomuraea Candida DSM 45086.

In a particular embodiment, the microorganism is selected from the group consisting of Tsukamurella sp. 1534 (Oulmi et al. J Bacterid. 2012 Oct; 194(19): 5482- 5483), Streptomyces aureus, preferably Streptomyces aureus DSM 41785, Streptomyces flavochromogenes, preferably Streptomyces flavochromogenes DSM 40541, Streptomyces natalensis, preferably Streptomyces natalensis DSM 40357, Nocardiopsis chromatogenes, preferably Nocardiopsis chromatogenes DSM 44844 and Nonomuraea Candida, preferably Nonomuraea Candida DSM 45086.

In another embodiment, , the microorganism is selected from the group consisting of Nocardia and Microbacterium bacteria, preferably from Nocardia terpenica, preferably Nocardia terpenica DSMZ 44935, Nocardia altamirensis, preferably Nocardia altamirensis DSMZ 44997, Microbacterium arborescens, preferably Microbacterium arborescens CIP 55.81T (Collection Institut Pasteur) or Microbacterium arborescens strain ND21 (NCBI BioSample: SAMN05211039), and Microbacterium sp. TS-1 (Uniprot taxon identifier : 1344956).

In a more particular embodiment, the microorganism is selected from the group consisting of Nocardia and Microbacterium bacteria, preferably from Nocardia terpenica, preferably Nocardia terpenica DSMZ 44935, Nocardia altamirensis, preferably Nocardia altamirensis DSMZ 44997, and Microbacterium arborescens, preferably Microbacterium arborescens CIP 55.81T (Collection Institut Pasteur). Optionally, the microorganism naturally producing said compound may be genetically modified to increase the production of said compound.

In a particular embodiment, the microorganism is a strain of Nocardia terpenica, preferably Nocardia terpenica DSMZ 44935, and the compound provided in step a) is of formula (VII)

(NOC1 compound of example 1)

In another particular embodiment, the microorganism is a strain of Nocardia altamirensis, preferably Nocardia altamirensis DSMZ 44997, and the compound provid

(VIII) wherein Y is a Cio saturated linear hydrocarbon chain, and the guanidine group is monomethylated, i.e. R2 is hydrogen and R3 is methyl, or vice versa (compound NOC 7 of example 1);

Y is a Cio saturated linear hydrocarbon chain, and the guanidine group is dimethylated, i.e. R2 and R3 are methyl (compound NOC 8 of example 1);

Y is a Cio unsaturated linear hydrocarbon chains comprising one double bond, preferably -(CH2)4-(CH2)_m-CH=CH-(CH2)_n-, wherein m and n are independently selected from 0 and integers from 1 to 4, and m+n = 4, and the guanidine group is monomethylated, i.e. R2 is hydrogen and R3 is methyl, or vice versa (compound NOC 4 of example 1);

Y is a Cio unsaturated linear hydrocarbon chains comprising one double bond, preferably -(CH2)4-(CH2)_m-CH=CH-(CH2)_n-, wherein m and n are independently selected from 0 and integers from 1 to 4, and m+n = 4, and the guanidine group is dimethylated, i.e. R2 and R3 are methyl (compound NOC 6 of example 1);

Y is a Cio unsaturated linear hydrocarbon chains comprising two double bonds, preferably -(CH₂)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)_P-, wherein m, n and p are independently selected from 0 and integers from 1 to 2, and m+n+p = 2, and the guanidine group is unmethylated, i.e. R2 and R3 are hydrogen (compound NOC 2 of example 1);

Y is a Cio unsaturated linear hydrocarbon chains comprising two double bonds, preferably -(CH₂)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)_P-, wherein m, n and p are independently selected from 0 and integers from 1 to 2, and m+n+p = 2, and the guanidine group is monomethylated, i.e. R2 is hydrogen and R3 is methyl, or vice versa (compound NOC 3 of example 1);

Y is a Cio unsaturated linear hydrocarbon chains comprising two double bonds, preferably -(CH2)4-(CH₂)m-CH=CH-(CH2)n-CH=CH-(CH2)_P-, wherein m, n and p are independently selected from 0 and integers from 1 to 2, and m+n+p = 2, and the guanidine group is dimethylated, i.e. R2 and R3 are methyl (compound NOC 5 of example 1);

Y is a C12 unsaturated linear hydrocarbon chain comprising three double bonds, preferably -(CH2)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)p-CH=CH-(CH₂)_q-, wherein m, n, p and q are independently selected from 0 and integers from 1 to 2, and m+n+p+q = 2, and more preferably -(CH₂)4-CH=CH-(CH₂)2-CH=CH-CH=CH-, and the guanidine group is monomethylated, i.e. R2 is hydrogen and R3 is methyl, or vice versa (compound NOC 9 of example 1); or Y is a C i2 unsaturated linear hydrocarbon chain comprising three double bonds, preferably -(CH2)4-(CH2)m-CH=CH-(CH2)n-CH=CH-(CH2)p-CH=CH-(CH₂)_q-, wherein m, n, p and q are independently selected from 0 and integers from 1 to 2, and m+n+p+q = 2, and more preferably -(CH₂)4-CH=CH-(CH₂)2-CH=CH-CH=CH-, and the guanidine group is dimethylated, i.e. R2 and R3 are methyl (compounds NOC 10 and NOC1 of example 1),

and a mixture thereof.

In a further particular embodiment, the microorganism is a strain of Microbacterium, preferably Microbacterium arborescens, more preferably Microbacterium arborescens CIP 55.8 IT, and the compound provided in step a) is of formul

wherein

Y is selected from the group consisting of -(CH₂) i₄- (compound A of example 2),

-(CH₂) i6 (compound C of example 2), and -(CH2)₄-(CH2)m-CH=CH-(CH2)_n-, wherein m and n are independently selected from 0 and integers from 1 to 10, and m+n = 10, preferably 2 < n (compound B of example 2). In another particular embodiment, the microorganism is Tsukamurella sp. 1534, and the antimicrobial compound(s) is(are) of formula (V) wherein Xi is A, X2 is V, X3 is S, X₄ is S and X₅ is G. In another particular embodiment, the microorganism is a strain of Streptomyces aureus, preferably Streptomyces aureus DSM 41785, or a strain of Streptomyces flavochromogenes, preferably Streptomyces flavochromogenes DSM 40541, and the antimicrobial compound(s) is(are) of formula (V) wherein Xi is A, X2 is G, X3 is S, X₄ is E and X5 is G.

In another particular embodiment, the microorganism is a strain of Streptomyces natalensis, preferably Streptomyces natalensis DSM 40357, and the antimicrobial compound(s) is(are) of formula (V) wherein Xi is A, X2 is T, X3 is S, X₄ is D and X5 is G.

In another particular embodiment, the microorganism is a strain of Nocardiopsis chromato genes, preferably Nocardiopsis chromatogenes DSM 44844, and the antimicrobial compound(s) is(are) of formula (V) wherein Xi is A, X2 is T, X3 is A, X₄ is D and X₅ is G.

In another particular embodiment, the microorganism is a strain of Nonomuraea Candida, preferably Nonomuraea Candida DSM 45086, and the antimicrobial compound(s) is(are) of formula (V) wherein Xi is A, X2 is A, X3 is S, X₄ is E and X5 is T.

In another embodiment, the compound provided in step a) is produced using a microorganism containing a gene encoding the precursor peptide. The gene may be endogenous to the microorganism, or mutated, or a heterologous gene introduced into said microorganism. In particular, the microorganism producing the compound may be a microorganism genetically modified to produce said compound, e.g. by introducing a heterologous gene or mutating the endogenous gene.

As used herein, the term "endogenous", with respect to a microorganism, refers to a genetic element or a protein naturally present in said microorganism. The term "heterologous", with respect to a microorganism, refers to a genetic element or a protein that is not naturally present in said microorganism.

In a particular embodiment, the compound provided in step a) is produced using a microorganism containing a gene encoding a precursor peptide comprising, or consisting of, a sequence selected from any of SEQ ID NOs: 5-7 and 18 to 23, preferably selected from any of SEQ ID NOs: 18 to 23, and more preferably from any of SEQ ID NOs: 18 to 22. In a more particular embodiment, the compound provided in step a) is produced using a microorganism containing a gene encoding a precursor peptide comprising, or consisting of, a sequence selected from anyone of SEQ ID NOs: 5-7.

In another embodiment, the compound provided in step a) is produced using a microorganism containing a gene encoding a precursor peptide comprising, or consisting of, a sequence selected from variants of any of SEQ ID NOs: 5-7, and 18 to 23, preferably selected from any of SEQ ID NOs: 18 to 23, and more preferably from any of SEQ ID NOs: 18 to 22. In a more particular embodiment, the compound provided in step a) is produced using a microorganism containing a gene encoding a precursor peptide comprising, or consisting of, a sequence selected from variants of anyone of SEQ ID NOs: 5-7.

Examples of such variants include, but are not limited to MID VTNI ADLHDID ATS GA AEL VGSISSNGC (SEQ ID NO: 8) and MID VTNI ADLHDID ATS GA AEL V ASLS S OGC (SEQ ID NO: 9), MS LEQLE ALP AS SE A AEM AGS IS SNGC (SEQ ID NO: 10), and MS LEQLE ALP AS SEA AEM A AS LS S OGC (SEQ ID NO: 11).

In particular, based on the sequences of the precursors (SEQ IP NO: 5 to 7 and 18 to 23) and the sequences of the leader sequences (SEQ IP NO: 1 to 3 and 12 to 17) of the compounds produced by Microbacterium arborescens, Nocardia terpenica, Nocardia altamirensis, Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida, and the general knowledge of the skilled person, genes encoding said precursors may be mutated in order to change the amino acid sequence of the core peptide. The required mutation may be easily determined by the skilled person based on codon usage.

Preferably, the leader sequence is not mutated, and more preferably corresponds to the leader sequence of the compound precursor naturally produced by the host microorganism.

Alternatively, a heterologous gene encoding the precursor may be introduced and expressed in a host microorganism.

In preferred embodiments, the host microorganism is naturally capable of producing a compound of formula (V) or (VI). Said microorganism is preferably selected from the group consisting of Nocardia, Microbacterium, Tsukamurella, Streptomyces, Nocardiopsis and Nonomuraea bacteria, more preferably from Microbacterium arborescens, Nocardia terpenica and Nocardia altamirensis, Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida.

In some preferred embodiments, said microorganism is preferably selected from Microbacterium and Nocardia bacteria, more preferably from Microbacterium arborescens, Nocardia terpenica and Nocardia altamirensis, and even more preferably is Microbacterium arborescens.

In some other embodiments, said microorganism is preferably selected from Tsukamurella, Streptomyces, Nocardiopsis and Nonomuraea bacteria, more preferably from Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida.

Suitable culture conditions such as medium, temperature and aeration parameters, may be easily defined by the skilled person according to the nature of the cultured microorganism.

Preferably, the compound provided in step a) is recovered from the culture supernatant. Extraction of said compound from the culture, and in particular from the supernatant, may be performed by any method known by the skilled person, for example by liquid-liquid extraction with an organic solvent such as butanol as illustrated in the experimental section.

The method may further comprise purifying said compound. The compound may be purified by any method known by the skilled person, for example using HPLC as illustrated in the experimental section, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. In step b) of the method of the invention, the lipophilic moiety Z is cleaved.

Preferably, this step is a deacylation removing/cleaving the lipophilic moiety Z attached to the core peptide, thereby providing a bicyclic peptide of the invention, in particular a bicyclic peptide of the invention of formula (I), (II), (III) or (IV) wherein B is hydrogen.

This deacylation reaction may be performed by any method known by the skilled person, i.e. chemically and/or enzymatically. In a preferred embodiment, this deacylation is enzymatically performed, preferably using the aculeacin-A deacylase (EC 3.5.1.70) produced by Actinoplanes utahensis NRRL 12052, an enzyme which is routinely used to cleave the fatty acid acyl group of lipopeptides (see e.g. Boeck et al. J Antibiot (Tokyo). 1988 Aug;41(8): 1085-92). Such deacylation is illustrated in example 3.

In this case, the compound provided in step a) is contacted with a deacylase or with a microorganism producing said deacylase, preferably with aculeacin-A deacylase of Actinoplanes utahensis NRRL 12052 or with Actinoplanes utahensis NRRL 12052.

Deacylation may be monitored by any analytical method known by the skilled person such as LC-UV or LC-MS analysis.

The method of the invention may further comprise isolating or purifying the bicyclic peptide of the invention. This step may be carried out using any method known by the skilled person such as HPLC as illustrated in the experimental section, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.

In another aspect, the present invention also relates to a method of producing a bicyclic peptide of the invention, said method comprising

providing a linear peptide comprising, or consisting of, the sequence B-X1-S-X2- X3-S-X4-X5-C (SEQ ID NO: 4), wherein B is a peptide chain of a size comprising from 1 to 30 amino acid residues, and Xi, X₂, X3, X₄ and X5 each represents an amino acid residue, preferably as defined above, and

Preferably, the method uses an enzymatic extract of a microorganism producing a bicyclic core peptide of formula (I) or (II), and/or producing a compound of formula (V), (VI), (X) or (XI).

In a particular embodiment, the linear peptide is a precursor peptide as defined above.

The linear peptide may be obtained by any method known by the skilled person in particular from a microorganism containing a gene encoding said peptide or using classical chemical synthesis (in solid phase or homogeneous liquid phase) and/or enzymatic synthesis. The linear peptide may then be contacted with an enzymatic extract of a microorganism producing a bicyclic core peptide of formula (I) or (II), or producing a compound of formula (V), (VI), (X) or (XI).

As used herein, the term "enzymatic extract" refers to a fraction comprising enzymes obtained from the microorganism biomass. The extract may be obtained by chemical, physical and/or enzymatic treatment(s). Preferably, the enzymatic extract is a partial or total extract of proteins obtained from the microorganism biomass. Such extract may be obtained using routine methods well known by the skilled person.

In the method of the invention, the enzyme or the enzymatic extract may be optionally immobilized on a support.

The linear peptide may be contacted with the extract until obtaining the bicyclic peptide of the invention.

The cyclization may be monitored by any analytical method known by the skilled person such as RMN, LC-UV and/or LC-MS analysis.

The method may further comprise isolating or purifying the bicyclic peptide of the invention as described above.

A further object of the invention also resides in a peptide comprising, or consisting of, the sequence B-X1-S-X2-X3-S-X4-X5-C (SEQ ID NO: 4), wherein B is a hydrogen, a peptide chain of a size of between 1 and 30 amino acid residues, or an a-amino-protecting group; and Xi, X₂, X3, X₄ and X5 are independently selected and each represents an amino acid, preferably as defined above.

Preferably, B is as defined above, and in particular is a peptide chain of a size comprised between 1 and 30 amino acid residues, more preferably is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MS LEQLE ALD AS SE A AEM A (SEQ ID NO: 1) MIDVTNIAELHELDSTSASAELV

(SEQ ID NO: 2), MID VTNIADLHDID ATS G A AELV (SEQ ID NO: 3),

MIDVTDINSLQAIESHSATSELL (SEQ ID NO: 12),

MDIADVMDLQGEEVVADGVELP (SEQ ID NO: 13),

MDLTNVMELQGTEIVADGVELP (SEQ ID NO: 14),

MDLANVMDLQGTEIVADGIELP (SEQ ID NO: 15),

MDLTNVIDLQGTEIVADGVELP (SEQ ID NO: 16) and

MTLEQLEALDASSEAAEMA (SEQ ID NO: 17), more preferably selected from SEQ ID NO 12 to 17, and in particular from SEQ ID NO 12 to 16. In particular embodiments, B is a peptide chain of a size comprised between 1 and 30 amino acid residues, more preferably is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MSLEQLEALDASSEAAEMA (SEQ ID NO: 1) MID VTNI AELHELD STS AS AELV (SEQ ID NO: 2) and MID VTNI ADLHDID ATS GA AEL V (SEQ ID NO: 3).

In a further aspect, the present invention also relates to a method of producing a lipolanthipeptide, preferably an antimicrobial lipolanthipeptide, comprising

a) providing a bicyclic peptide of the invention, and

b) adding a lipophilic moiety to said peptide.

In embodiments wherein B is a peptide chain or an a-amino-protecting group as defined above, the method further comprises before step b) removing/cleaving said peptide chain or protective group. This step may be carried out using any method described above.

Step b) may be performed e.g. chemically or enzymatically.

Preferably, step b) is an acylation, more preferably a chemical acylation. In particular, a halogenated lipophilic moiety, preferably a chlorinated lipophilic moiety, may be added to the bicyclic peptide, in the presence of an acylation catalyst, preferably selected from pyridine or 4-dialkylaminopyridines such as 4-dimethylaminopyridine. Such acylation reaction is illustrated in example 3.

The lipophilic moiety may be a naturally occurring moiety, a modified naturally occurring moiety or a synthetic one.

In a particular embodiment, the lipophilic moiety is represented as R4-W-L- wherein

L is a bifunctional linker, preferably selected from the group consisting of -C(=0)- , -SO2-, -C(=S)-, -OC(=S)-, -PO-, -OPO-, -OC(=0)-, -NHC(=0)- and -NHC(=S),

W is a saturated or unsaturated linear hydrocarbon chain, optionally substituted and/or interrupted, and

P4 is selected from the group consisting of hydrogen and a basic group. The bicyclic core peptide and the lipophilic moiety are linked via a bifunctional linker. As used herein, the term "bifunctional linker " refers to any chemical group being able to connect two chemical groups, and in particular being able to covalently connect at the same time (i) a hydrocarbon chain and (ii) an amino group.

Typically, L comprises 1 to 25 atoms, preferably 1 to 10 atoms, and at least one heteroatom selected from O, S and P.

Preferably, L is selected from the group consisting of -C(=0)-, -SO2-, -C(=S)-, - 0-C(=S)-, -NH-C(=S)-, -PO-, -OPO-, -OC(=0)- and -NHC(=0)-, more preferably from the group consisting of -C(=0)-, -SO2-, -C(=S)-, -OC(=0)- and -NHC(=0)-.

In preferred embodiments, L is -C(=0)-.

W may be a saturated or unsaturated linear hydrocarbon chain, optionally substituted and/or interrupted, preferably a C6-C20 saturated or unsaturated linear hydrocarbon chain, said chain being optionally (i) interrupted by one or several heteroatoms independently selected from N, S and O, and/or (ii) interrupted by one or several groups independently selected from a phenyl group and a 5 or 6-membered-ring heterocycle, said phenyl group or heterocycle being optionally substituted, for example, by one or several groups independently selected from C1-C3 alkyl groups, -OH and C1-C3 alkoxy groups, and/or (iii) substituted by one or several groups independently selected from C1-C3 alkyl groups, halogens, -OH, methoxy or acetoxy.

In particular embodiments, W is selected from saturated or unsaturated linear hydrocarbon chains as defined above for Y.

Furthermore, W may be substituted by one or several groups independently selected from C1-C3 alkyl groups, halogens, -OH, methoxy and acetoxy.

As used herein, C1-C3 alkyl groups encompass methyl, ethyl, propyl and isopropyl. Halogens may be selected from F, CI and Br.

In a particular, the saturated or unsaturated linear hydrocarbon chain may be substituted by one group selected from C1-C3 alkyl groups, halogens, -OH, methoxy and acetoxy, preferably -OH.

In further embodiments, W is a saturated or unsaturated linear hydrocarbon chain, in particular Y as described above, interrupted by one or several heteroatoms independently selected, preferably from N, S and O, and/or by one or several groups independently selected from a phenyl group and a 5 or 6-membered-ring heterocycle, said phenyl group or heterocycle being optionally substituted, for example, by one or several groups independently selected from C1-C3 alkyl groups, -OH and C1-C3 alkoxy groups.

As used herein, the term "heteroatom" refers to any atom that is not carbon or hydrogen. In preferred embodiments, this term refers to N, S, or O.

The term "heterocycle", as used herein, refers to 5- or 6-membered heterocyclic ring systems comprising one or more heteroatoms, preferably 1 or 2 endocyclic heteroatoms. Preferably, they are monocyclic systems. They may be aromatic or not. Examples of 5- or 6-membered-ring heterocycles include furan, pyrrole, thiophene, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, triazole, pyridine, pyrane, piperidine, dioxane, pyrazine and pyrimidine.

In a particular embodiment, the hydrocarbon chain as described above is interrupted by one or several heterocycles, preferably by one, two or three heterocycles. In such embodiment, the heterocycle(s) may be inserted in the chain in one of the follow

wherein X, Y, W and Z are independently selected from carbon and nitrogen, and

wherein X, W and Z are independently selected from carbon, nitrogen, sulfur and oxygen, and V and Y are independently selected from carbon and nitrogen,

with the proviso that the 5-membered heterocycle is inserted in the chain in one of the following configurations:

when V is nitrogen.

In embodiments wherein the chain is interrupted by several heterocycles, the configuration of each heterocycle may be independently selected from these configurations.

In another particular embodiment, the hydrocarbon chain as described above is interrupted by one or several phenyl groups, preferably by one, two or three phenyl groups. In such embodiment, phenyl groups may be inserted in the chain in one of the follow

In embodiments wherein the chain is interrupted by several phenyl groups, the configuration of each phenyl group may be independently selected from these configurations.

In another embodiment, the chain is interrupted by one or two phenyl groups and one or two heterocycles.

The phenyl groups and/or heterocycles may be jointed, so as to form for example naphthalene, benzofuran, indole and/or quinoline groups, or separated by one or several carbons of the hydrocarbon chain.

The interrupted saturated or unsaturated linear hydrocarbon chain may be any saturated or unsaturated linear hydrocarbon chain Y or W as described above, including substituted and unsubstitued saturated or unsaturated linear hydrocarbon chain. selected from the group consisting of hydrogen and a basic group. As used herein, the term "basic group" refers to an organic group which is a proton acceptor. Illustrative basic groups are primary, secondary, tertiary acyclic or cyclic amines, amidines, guanidines.

In an embodiment, R4 is selected from the group consisting of hydrogen and a basic

with R5 and R₆ being independently selected from hydrogen, C1-C3 alkyl groups and -C(=0)R7, and R7 being a C1-C3 alkyl group. In a preferred embodiment, R₄ is selected from the group consisting of hydrogen and a b

with R5 and R₆ being independently selected from hydrogen, C1-C3 alkyl groups and -C(=0)R7, and R7 being a C1-C3 alkyl group.

It should be noted that tautomeric forms of the roups described above are also

ntemplated. As illustration, as used herein,

also encompass

and also encompasses

In a specific embodiment, R₄ is Ri as defined above.

In another aspect, the present invention also relates to an expression cassette comprising a coding region encoding a linear peptide comprising, or consisting of, the sequence B-Xi-S-X₂-X₃-S-X₄-X₅-C (SEQ ID NO: 4), wherein Xi, X₂, X₃, X₄ and X₅ are independently selected and each represents an amino acid; and B is selected from the group consisting of hydrogen and a peptide chain of a size comprised between 1 and 30 amino acid residues, said coding region being operably linked to one or more control sequences that direct the expression of said coding region in a suitable host cell under conditions compatible with the control sequences.

Xi, X₂, X3, X₄ and X5 may be selected as defined above for the bicyclic peptide of the invention.

Preferably, B is as described above, and in particular a peptide chain of a size comprised between 1 and 30 amino acid residues, more preferably is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MSLEQLEALDAS SEAAEM A (SEQ ID NO: 1) MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2), MID VTNIADLHDID ATS G A AELV (SEQ ID NO: 3), MIDVTDINSLQAIESHSATSELL (SEQ ID NO: 12),

MDIADVMDLQGEEVVADGVELP (SEQ ID NO: 13),

MDLTNVMELQGTEIVADGVELP (SEQ ID NO: 14),

MDLANVMDLQGTEIVADGIELP (SEQ ID NO: 15), MDLTNVIDLQGTEIVADGVELP (SEQ ID NO: 16) and MTLEQLEALDASSEAAEMA (SEQ ID NO: 17), more preferably selected from SEQ ID NO 12 to 17, and in particular from SEQ ID NO 12 to 16. In a particular embodiment, B is a peptide chain of a size comprised between 1 and 30 amino acid residues, more preferably is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MSLEQLEALDASSEAAEMA (SEQ ID NO: 1) MID VTNI AELHELD STS AS AELV (SEQ ID NO: 2) and MID VTNI ADLHDID ATS GA AEL V (SEQ ID NO: 3).

The term "expression cassette" denotes a nucleic acid construct comprising a coding region, i.e. one or several genes, and a regulatory region, i.e. comprising one or more control sequences, operably linked. Optionally, the expression cassette may comprise several coding regions operably linked to several regulatory regions.

The term "control sequences" means nucleic acid sequences necessary for expression of a coding region. Control sequences may be endogenous or heterologous. Well-known control sequences and currently used by the person skilled in the art will be preferred. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. Preferably, the control sequences include a promoter and a transcription terminator.

The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, in such a way that the control sequence directs expression of the coding region.

The control sequence may include a promoter that is recognized by a host cell for expression of the coding region. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be an endogenous or heterologous promoter. The promoter may be a strong, weak, constitutive or inducible promoter.

Optionally, the expression cassette may also comprise a selectable marker that permits easy selection of recombinant host cells. Typically, the selectable marker is a gene encoding antibiotic resistance or conferring autotrophy.

The expression cassette of the invention may be used directly to transform a host cell and enable the expression of the coding region. Preferably, the expression cassette, or a part thereof comprising the coding region, is inserted in the genome of the host cell. In a particular embodiment, the expression cassette is integrated in the genome of the host cell, preferably to replace the endogenous gene encoding the precursor of the bicyclic peptide of the invention.

The present invention also relates to an expression vector comprising an expression cassette according to the invention.

As used herein, the term "expression vector" means a DNA or RNA molecule that comprises an expression cassette. Preferably, the expression vector is a linear or circular double stranded DNA molecule.

The expression vector of the invention may be used to transform a host cell and enable the expression of the coding region in said cell. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini- chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Preferably, the vector, or a part thereof comprising the coding region, e.g. the expression cassette of the invention, is inserted in the genome of the host cell, e.g. to replace the endogenous gene encoding the precursor of the bicyclic peptide of the invention.

The vector preferably comprises one or more selectable markers that permit easy selection of host cells comprising the vector. A selectable marker is a gene the product of which provides for antibiotic resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like.

The vector preferably comprises an element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. When integration into the host cell genome occurs, integration of the sequences into the genome may rely on homologous or non-homologous recombination. In one hand, the vector may contain additional polynucleotides for directing integration by homologous recombination at a precise location into the genome of the host cell. These additional polynucleotides may be any sequence that is homologous with the target sequence in the genome of the host cell. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.

The methods for selecting these elements according to the host cell in which expression is desired, are well known to one of skill in the art. The vectors may be constructed by the classical techniques of molecular biology, well known to one of skill in the art.

The present invention further relates to the use of an expression cassette or an expression vector according to the invention to transform, transfect or transduce a host cell. The present invention also relates to a host cell comprising an expression cassette or an expression vector according to the invention.

The host cell may be transformed, transfected or transduced in a transient or stable manner. An expression cassette or vector of the invention is introduced into a host cell so that the cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.

Preferably, the host cell is a microorganism, in particular a bacterium or a fungus. More preferably, the host cell is a bacterium. In some embodiments, the host cell belongs to Microbacterium, Nocardia, Tsukamurella, Streptomyces, Nocardiopsis or Nonomuraea genus, preferably Microbacterium or Nocardia genus. In some particular embodiments, the host cell is selected from the group consisting of Microbacterium arborescens, Nocardia terpenica, Nocardia altamirensis, Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida, preferably from the group consisting of Microbacterium arborescens, Nocardia terpenica and Nocardia altamirensis. In some other embodiments, the host cell is Escherichia coli.

The expression cassette or expression vector according to the invention may be introduced into the host cell by any method known by the skilled person, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation or liposome-mediated transformation.

Optionally, more than one copy of a nucleic acid, cassette or vector of the present invention may be inserted into the host cell.

In a specific aspect, the present invention relates to a microorganism genetically modified to express a heterologous gene encoding a linear peptide comprising, or consisting of, the sequence B-X1-S-X2-X3-S-X4-X5-C (SEQ ID NO: 4), wherein Xi, X₂, X3, X₄ and X5 are independently selected and each represents an amino acid, and B is selected from the group consisting of hydrogen and a peptide chain of a size comprised between 1 and 30 amino acid residues.

Preferably, B is as defined above, and in particular a peptide chain of a size comprised between 1 and 30 amino acid residues, more preferably is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MS LEQLE ALD AS SE A AEM A (SEQ ID NO: 1) MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2), MID VTNIADLHDID ATS G A AELV (SEQ ID NO: 3),

MIDVTDINSLQAIESHSATSELL (SEQ ID NO: 12),

MDIADVMDLQGEEVVADGVELP (SEQ ID NO: 13),

MDLTNVMELQGTEIVADGVELP (SEQ ID NO: 14), MDLANVMDLQGTEIVADGIELP (SEQ ID NO: 15),

MDLTNVIDLQGTEIVADGVELP (SEQ ID NO: 16) and MTLEQLEALDASSEAAEMA (SEQ ID NO: 17), more preferably selected from SEQ ID NO 12 to 17, and in particular from SEQ ID NO 12 to 16. In a particular embodiment, B is a peptide chain of a size comprised between 1 and 30 amino acid residues, more preferably is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MSLEQLEALDASSEAAEMA (SEQ ID NO: 1) MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2) and MID VTNIADLHDID ATS GA AEL V (SEQ ID NO: 3). Preferably, the microorganism is a bacterium or a fungus. More preferably, the microorganism is a bacterium. In some embodiments, the host cell belongs to Microbacterium, Nocardia, Tsukamurella, Streptomyces, Nocardiopsis or Nonomuraea genus, preferably Microbacterium or Nocardia genus. In some particular embodiments, the host cell is selected from the group consisting of Microbacterium arborescens, Nocardia terpenica, Nocardia altamirensis, Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida, preferably from the group consisting of Microbacterium arborescens, Nocardia terpenica and Nocardia altamirensis. In some other embodiments, the host cell is Escherichia coli.

The expression cassette or expression vector according to the invention may be used to genetically modify the microorganism, e.g. by transformation, transfection or transduction.

In preferred embodiments, the microorganism is naturally capable of producing a lipolanthipeptide of any formula (V) to (XI).

Said microorganism is preferably selected from the group consisting of Nocardia, Microbacterium, Tsukamurella, Streptomyces, Nocardiopsis and Nonomuraea bacteria, more preferably from Microbacterium arborescens, Nocardia terpenica and Nocardia altamirensis, Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida.

In a particular embodiment, the microorganism is selected from Microbacterium and Nocardia bacteria, more preferably from Microbacterium arborescens, Nocardia terpenica and Nocardia altamirensis.

In a specific embodiment, the microorganism is a Microbacterium bacterium, more preferably Microbacterium arborescens.

In another embodiment, the microorganism is selected from Tsukamurella, Streptomyces, Nocardiopsis and Nonomuraea bacteria, more preferably from Tsukamurella sp. 1534, Streptomyces aureus, Streptomyces flavochromogenes, Streptomyces natalensis, Nocardiopsis chromatogenes and Nonomuraea Candida.

In some particular embodiments, - the host cell belongs to Microbacterium genus, preferably is Microbacterium arborescens, and B comprises, or consists of, a sequence selected from SEQ ID NO: 1 and 17, and variants thereof, preferably comprises or consists of, SEQ ID NO: 1 or 17; or

- the host cell belongs to Nocardia genus, preferably is Nocardia terpenica or Nocardia altamirensis, and B comprises, or consists of, a sequence selected from SEQ

ID NO: 2 and 3, and variants thereof, preferably comprises or consists of, SEQ ID NO: 2 or 3; or

- the host cell belongs to Tsukamurella genus, preferably is Tsukamurella sp. 1534, and B comprises, or consists of, a sequence selected from SEQ ID NO: 12, and variants thereof, preferably comprises or consists of, SEQ ID NO: 12; or

- the host cell belongs to Streptomyces genus, preferably is selected from Streptomyces aureus, Streptomyces flavochromogenes and Streptomyces natalensis, and B comprises, or consists of, a sequence selected from SEQ ID NO: 14 and 16, and variants thereof, preferably comprises or consists of, SEQ ID NO: 14 or 16, or

- the host cell belongs to Nocardiopsis genus, preferably is Nocardiopsis chromato genes, and B comprises, or consists of, a sequence selected from SEQ ID NO: 13, and variants thereof, preferably comprises or consists of, SEQ ID NO: 13, or

- the host cell belongs to Nonomuraea genus, preferably is Nonomuraea Candida, and B comprises, or consists of, a sequence selected from SEQ ID NO: 15, and variants thereof, preferably comprises or consists of, SEQ ID NO: 15.

The present invention also relates to the use of said genetically modified microorganism of the invention to produce a bicyclic peptide of the invention or a lipolanthipeptide of any formula (V) to (XI).

Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting. EXAMPLES

Example 1

Preparation of culture medium for production of lipolanthipeptide from

Nocardia bacteria

GYM medium

The composition of the GYM liquid medium was as follows: 4 g Glucose, Yeast extract, 10 g Malt extract and 1000 ml distilled water.

The 10% glucose and 3M KOH solutions were prepared separately.

The 10% slucose (100 ml)

10 g of powder, distilled water qsp 100 mL

Sterilization at 110°C for 30 minutes

3M KOH (1000 ml)

MM = 56.11 g/mol

Purity: 85%

56.11 * 0.85 = 47.6 g/mol

Weigh 143.08 g of powder for a qsp of 1 L with distilled water

Autoclave at 121 °C for 20 minutes

GYM medium liquid ( 1000 ml)

4 g yeast extract

10 g malt extract

Sterilization at 121°C for 20 minutes

40 ml sterile 10% glucose: final concentration 0.4 % (final concentration 4 g/L) Adjust pH to 7.2 using sterile KOH

GYM medium agar (1000 ml)

Add CaCOs 2.0 g/L and Agar 15.0 g/L Culture of Nocardia terpernica or Nocardia altamirensis

- Preculture (PL)

A 500 ml flask containing as final volume 100 ml GYM medium was inoculated with a colony of the primary Nocardia terpenica (DSMZ 44935) or Nocardia altamirensis (DSMZ 44997) strain and incubated at 30°C for 24 h with stirring at 240 rotations per minute (rpm). Optical density (OD) at 600 nm was then measured by a spectrophotometer until the Nocardia terpenica or Nocardia altamirensis strain was at the beginning /middle of its exponential growth phase (1 <OD at 600 nm <3)

The purity of the pre-culture was monitored by seeding on GYM agar. The plates were incubated at 30°C for 48 h.

- Cultures in Erlenmeyer flasks

A 5000 ml flask, containing as a final volume 1000 ml GYM medium was inoculated with the 100 ml of pre-culture (PI) and incubated at 30°C for 96 hours with stirring at 240 rpm. Initial OD at 600 nm ranged between 0.1 and 0.3.

Purity of fermentation was monitored at the end of 96 hours by seeding a GYM agar. The plates were incubated at 30°C for 48 h.

The culture was centrifuged to 10,000 g for 45 min at 25 °C.

The supernatant was recovered and kept at 4°C

Extraction and analysis of the compounds having antimicrobial activity from Nocardia altamirensis

Extraction of the compounds having antimicrobial activity from the supernatant was carried out by liquid-liquid extraction with butanol. Butanol was concentrated to dryness in a rotary evaporator at 50°C to give the crude extract.

Compounds having antimicrobial activity were analyzed in the crude extract. LC- MS/MS analysis of the crude extract is presented in figures 1 and 2 and Table 1. LC-MS conditions

Phenomenex Gemini NX, 5μ, C18, 110 A, 150 X 2 mm Gradient HPLC

Column Gemini C18 5μιη, 100A 150 x 2mm

Solvent A: H20+0.1% formic acid

Solvent B: ACN + 0.1% formic acid

Timet min) Flow Rate(mL/min) %A %B

1. Initial 0.500 95.0 5.0

2. 2.00 0.500 95.0 5.0

3. 14.00 0.500 0.0 100.0

4. 15.00 0.500 0.0 100.0

5. 17.00 0.500 95.0 5.0

MS/MS conditions MS

Polarity: ES+

Capillary (kV): 3.0000

Source Temperature (°C): 150

Sampling Cone: 30.0000

Source Offset: 90.0000

Source Gas Flow (mL/min): 0.00

Desolvation Temperature (°C): 300

Cone Gas How (L/Hr): 0.0

Desolvation Gas Flow (L/Hr): 600.0

Nebuliser Gas Flow (Bar): 3.0

Ion Energy: 1.0

Acquisition mass range

Start mass: 100.000

End mass: 2000.000

Function Parameters - Function 1 - TOF FAST DDA FUNCTION

[MS SURVEY]

Survey Start Mass: 100.0000 Survey End Mass: 2000.0000

Switch to MS/MS when Intensity rising above threshold

Intensity Threshold: 5000.0

Survey Scan Time: 0.2

Survey InterScan Time: 0.01

Survey Data Format: Continuum

Analyser: Resolution Mode

MS/MS (automatic acquisition of 4 precursor ions)

MSMS Start Mass: 100.0

MSMS End Mass: 2000.0

Number of precusors: 4

MSMS to MS Switch Criteria: TIC rising above threshold

Switchback threshold: 50000.0

MSMS Switch After Time (sec): 0.2

MSMS Scan Time (sec): 0.10

MSMS InterScan Time (sec): 0.01

MSMS Data Format: Continuum

[COLLISION ENERGY]

Trap MSMS Collision Energy Ramp Low Mass (Da): 100.0

Trap MSMS Collision Energy Ramp High Mass (Da): 2000.0

Trap MSMS Collision Energy Ramp LM Start (eV): 10.0

Trap MSMS Collision Energy Ramp LM End (eV): 20.0

Trap MSMS Collision Energy Ramp HM Start (eV): 70.0

Trap MSMS Collision Energy Ramp HM End (eV): 100.0

[TRANFER COLLISION ENERGY]

Using MSMS Auto Trap Collision Energy (eV): 2.000000

[CONE VOLTAGE]

Cone (V): 40.0

The antimicrobial compounds isolated from Nocardia altamirensis have the following formula (VIII)

O (VIII) R2, R3 and Y being defined in Table 1 below. Table 1 : Compounds from Nocardia altamirensis

Molecular Molecular

Name Time Retention HR-(M+H)⁺ weight formula

NOC2 5.64 876 C38H60N12O10S 877.4365

NOC3 5.69 890 C39H62N12O10S 891.4538

NOC4 5.74 892 C39H64N12O10S 893.4629

NOC5 5.74 904 C40H64N12O10S 905.4678

NOC6 5.81 906 C40H66N12O10S 907.4797

NOC7 5.89 894 C39H66N12O10S 895.4803

NOC8 5.95 908 C40H68N12O10S 909.4958

NOC9 6.11 916 C41H64N12O10S 917.4649

NOC10 6.13 930 C42H65N12O10S 931.4836

Y

(unsubstituted and uninterrupted

Name R2 / R3 linear hydrocarbon chain)

Number of

Chain lenght

double bonds

NOC2 Hydrogen CIO 2

hydrogen and

NOC3 CIO 2

methyl hydrogen and

NOC4 CIO 1

methyl

NOC5 methyl CIO 2

NOC6 methyl CIO 1

hydrogen and

NOC7 CIO 0

methyl

NOC8 methyl CIO 0

hydrogen and

NOC9 C12 3

methyl

NOC10 methyl C12 3

Extraction, purification and structure of the antimicrobial compound isolated from Nocardia terpenica (NOC1)

Extraction of the compounds having antimicrobial activity from the supernatant was carried out by liquid-liquid extraction with butanol. Crude extract was purified by taking 150 mg in a mixture of H2O/ACN/DMSO 1/1/1 (v/v/v). The sample was manually loaded (5.0 mL) into the injection system of the semi-preparative HPLC manufactured by Waters. The column used was a CI 8 (5 microns, 150 x 10 mm, Gemini, Phenomenex). Elution was performed at a flow rate of 7 mL/min according to the gradient shown in Table 2 below.

Table 2 : Elution as a function of respective concentrations of buffers A and B

The peak corresponding to compound NOC1 was collected at 10.9 min.

The obtained compound was analyzed by LC-MS/MS analysis (figure 3 and figure

4) and by NMR 600 Mhz (cf. Figures 10 to 13). According to these analysis, the antimicrobial compound isolated from Nocardia terpenica has the following formula (VII)

(VII)

Antibacterial activities of the compound NOC1

The measures of activities were conducted on compound NOC 1 from Nocardia terpenica following the protocol recommended by the Clinical and Laboratory Standards Institute (CLSI) - Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS): Methods for Dilution Antibacterial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Tenth Edition (2015). Clinical and Laboratory Standards Institute Document M07-A10. The activities are illustrated in tables 3 and 4 hereafter.

Table 3: MIC for compound NOC1 against different strains of Staphylococcus

Minimal Inhibitory

Strain

Concentration (MIC)

S. aureus - ATCC 13709 (Fully susceptible) 2

S. aureus - ATCC 1683 (Methicillin resistant) 16

S. aureus - ATCC 25923 2

S. aureus - USA300 60 Table 4: Extended antimicrobial activities of compound NOCl

Strain ID Strain Characterized MIC

Resistance ^g/mL)

Gram-positive

Aerobe :

ATCC 13709 Staphylococcus aureus Methicillin sensitive 2

ATCC1683 Staphylococcus aureus Methicillin resistant 16

ATCC25923 Staphylococcus aureus Methicillin sensitive 2

USA300 Staphylococcus aureus Methicillin resistant 60

ATCC29212 Enterococcus faecalis Vancomycin sensitive 160

ATCC700802 Enterococcus faecalis Vancomycin resistant 16

(gene vanB)

ATCC 19434 Enterococcus faecium Vancomycin sensitive 8

ATCC51858 Enterococcus faecium Vancomycin resistant 16

(gene vanB)

ATCC51559 Enterococcus faecium Vancomycin resistant 16

(gene vanA)

ATCC6633 Bacillus subtilis 2

Fungi :

ATCC 10231 Candida albicans 3

DSMZ5784 Candida parapsilosis 24

DSMZ6128 Candida krusei 12

DSMZ6425 Candida glabrata 12

DSMZ6972 Cryptococcus neoformans 24

DSMZ11953 Candida tropicalis 6 Example 2

Preparation of culture medium for production of lipolanthipeptide from Microbacterium bacteria

5

YPG (peptone, glucose, yeast extract) medium

The composition of the YPG medium is as follows: glucose, 1 g/L; peptone, 10 g/L; yeast extract, 5 g/L; MOPS (3- (N-morpholino)propansulfonic acid) 150 mM

The 10% glucose, 2M MOPS and 3M KOH solutions are prepared separately.

YPG medium

• 10 g/L of peptone

• 5 g/L yeast extract

Sterilization at 121°C for 20 minutes

Addition of sterile 10% glucose: final concentration 0.1% (final concentration 1

Addition of sterile MOPS (final concentration 150 mM)

Adjust pH to 7.2 using sterile KOH or sterile KC1 depending on the initial pH.

20 Culture of Microbacterium arborescens CIP 55.8 IT.

Pre-culture (PL)

A 500 ml flask containing as final volume 100 ml YPG medium was inoculated with a colony of the primary Microbacterium arborescens strain bank and incubated at 25 30°C for 24 h with stirring at 160 rotations per minute (rpm). Optical density (OD) at 600 nm was then measured by a spectrophotometer until the Microbacterium arborescens strain was at the beginning /middle of its exponential growth phase (1 <OD at 600 nm <3).

The purity of the pre-culture was monitored by seeding on YPG agar. The plates 30 were incubated at 30°C for 48 h. - Cultures in Erlenmeyer flasks

A 5000 ml flask, containing as a final volume 1000 ml YPG medium was inoculated with the 100 ml of pre-culture (PI) and incubated at 30°C for 96 hours with stirring at 160 rpm. Initial OD at 600 nm ranged between 0.1 and 0.3.

Purity of fermentation was monitored at the end of 96 hours by seeding a YPG agar. The plates were incubated at 30°C for 48 h.

The culture was centrifuged to 10,000 g for 45 min at 25 °C.

The supernatant was recovered and kept at 4°C Extraction of lipolanthipeptide

Extraction of the compounds having antimicrobial activity from the supernatant was carried out by liquid-liquid extraction in contact with a mixture of dichloromethane /methanol in a 80: 20 ratio. The operation is carried out 5 times using the collected supernatant. The solvent was concentrated to a final volume of 20 ml in a rotary evaporator at 50°C, 7 mbar, 160 rpm. A precipate was formed, the supernatant was taken off and the precipitate (brown) (PRE1) was redissolved in methanol and the solvent was evaporated under vacuum.

PRE1 was washed several times with dichloromethane then with dichloromethane / Methanol (99/1) to obtain precipitate 2 (yellow) (PRE2).

Purification by preparative HPLC

PRE2 was purified by taking 150 mg in a mixture of DMSO, H2O, acetonitrile 1/1/1 (v/v/v). The sample was manually loaded (1.5 mL) into the injection system of the semi-preparative HPLC manufactured by Waters. The column used was a C18 (5 microns, 150 x 21 mm, Gemini, Phenomenex). Elution was performed at a flow rate of 15 mL/min according to the gradient shown in Table 1 below:

Table 5: Elution as a function of respective concentrations of buffers A and B

Time (min) Buffer A (H₂0) Buffer B (Acetonitrile +

0.1% formic acid)

0 100 0

2 100 0

17 50 50 19 0 100

23 0 100

25 100 0

30 100 0

The three peaks corresponding to compounds A, B and C were collected at 15.1 min, 15.8 min and 16.3 min respectively.

The obtained compounds were analyzed by MALDI-TOF mass spectrometry and by NMR.

The general structure of compounds A, B and C was as follow:

wherein

for compound A : Y is -(CH₂) i4-,

for compound B : Y is -(CH2)4-(CH2)m-CH=CH-(CH2)_n-, wherein m and n are independently selected from 0 and integers from 1 to 10, and m+n = 10; and

for compound C : Y is -(CH₂)i₆. Antimicrobial activities of compounds A, B and C

The measures of activities were conducted on compounds A, B and C, following the protocol recommended by the Clinical and Laboratory Standards Institute (CLSI) - Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS): Methods for Dilution Antibacterial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Tenth Edition (2015). Clinical and Laboratory Standards Institute Document M07-A10.

Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard - Eighth Edition (2012). Clinical and Laboratory Standards Institute Document Ml 1-A8.

Antimycobacterial activity was determined as described in Journal of Clinical Microbiology (2009, 47: 1773-1780) by Springer et al. Quantitative drug susceptibility testing of Mycobacterium tuberculosis by use of MGIT 960 and EpiCenter Instrumentation.

The activities are illustrated in tables 6 and 7 hereafter.

Table 6: MIC for compounds A, B and C against different strains of Staphylococcus aureus

Table 7: Extended antimicrobial activities of compound C

Strain ID Strain Characterized MIC

Resistance ^g/mL)

Gram-positive Aerobe :

ATCC 13709 Staphylococcus aureus Methicillin sensitive < 0.04

ATCC1683 Staphylococcus aureus Methicillin resistant < 0.3

37361192 Staphylococcus epidermidis Methicillin sensitive < 0.25

31435861 Staphylococcus epidermidis Methicillin resistant < 0.25

31432663 Streptococcus agalactiae < 0.25 37352281 Streptococcus pyogenes < 0.5

39050149 Streptococcus mitis < 0.25

39151368 Streptococcus oralis < 0.25

R119 (R6 like) Streptococcus pneumoniae Penicillin sensitive < 0.125

6883 Streptococcus pneumoniae Penicillin resistant < 0.125

ATCC1858 Enterococcus faecium Vancomycin sensitive < 0.5

31152980 Enterococcus faecium Van A resistant < 0.25

31430797 Enterococcus faecium Van B resistant < 0.25

Gram-positive Anaerobe :

ATCC 700057 Clostridium difficile < 0.25

1201 Propionibacterium acnes < 0.06

Mycobacteria :

H37Rv M. tuberculosis <1

Example 3

The lipophilic moiety of compound A described in example 2 was eliminated by enzymatic deacylation and replaced by stearoyl chain.

Preparation of the bicyclic core peptide

A. Fermentation of Actinoplanes utahensis

A stock culture of Actinoplanes utahensis NRRL 12052 is prepared and maintained on an agar slant. The medium used to prepare the slant is selected from one of the following:

TSB MEDIUM: Tryptic Soy Broth Formula Per Liter Purified Water: Tryptone (Pancreatic Digest of Casein) 17.0 g, Soytone (Peptic Digest of Soybean Meal) 3.0 g, Glucose (=Dextrose) 2.5 g, Sodium Chloride 5.0 g, Dipotassium Hydrogen Phosphate 2.5 g, pH 7.3 ± 0.2.

The pH of the medium was about 7.0 after sterilization by autoclaving at 121° C. for 20 minutes. The slant was inoculated with Actinoplanes utahensis NRRL 12052, and the inoculated slant was incubated at 30° C. for about 8 to 10 days. The slant was used to inoculate 100 ml of TSB MEDIUM.

TSB MEDIUM was incubated in a 500-ml baffle Erlenmeyer flask at 30° C for 5 about 72 hours on a shaker at 240 RPM.

In order to provide a larger volume of inoculum, 100 ml of the incubated TSB medium 100 ml was used to inoculate 900 ml of TSB medium having the same composition as the TSB medium 100 ml.

TSB MEDIUM 1000 ml was incubated in a 5000 ml baffle Erlenmeyer flask at 10 30° C for about 120 hours on a shaker at 240 RPM.

B. Concentration of the Enzyme

Whole fermentation broth was centrifugated for 45 min at 10000 g. The supernatant (100 ml) was concentrated with Centricon Plus-70 Centrifugal Filter, 15 Ultracel-PL Membrane, 30kDa from Millipore. The filtrate (5 ml) thus obtained was collected.

C. Deacylation of compound A

Reaction mixture :

20 0.85 mg of purified compound A was added to 0.65 ml of distilled water to obtain a concentration of 1.3 mg/ml

0.35 ml of PBS 0.1 M

0.25 ml of concentrated enzyme as described in section B

Temperature of 45 °C under magnetic agitation

5 Deacylation of compound A was monitored by HPLC analysis with a UV monitor at 254 nm.

The column used was a CI 8 (5 microns, 150 x 2 mm, Gemini, Phenomenex). Elution was performed at a flow rate of 0.5 mL/min according to the gradient shown in Table 8 below: Table 8: Elution as a function of respective concentrations of buffers A and B

LC-UV and LC -MS/MS analysis of the reaction mixture is presented in figures 5 to 8. D. Isolation of the core peptide

Reaction mixture was briefly centrifugated (8000 g - 5 min) and the core peptide was purified by taking 1 ml of the reaction mixture. The sample was manually loaded into the injection system of the semi-preparative HPLC manufactured by Waters. The column used was a CI 8 (5 microns, 150 x 10 mm, Gemini, Phenomenex). Elution was performed at a flow rate of 7 mL/min according to the gradient shown in Table 9 below:

Table 9 : Elution as a function of respective concentrations of buffers A and B

The peak corresponding to the core peptide was collected at 5.8 min, concentrated under vacuum to remove the acetonitrile to give about 100 μg of purified core peptide.

E. Acylation o f the core peptide with stearoyl chloride

To a solution of 600 μg of purified core peptide prepared as above in DMF (450μί) was added successively pyridine (450μί), a few amount of 4- dimethylaminopyridine and stearoyl chloride (300 μί). The reaction mixture was stirred at room temperature for 2 h and then concentrated to dryness to give a mixture of the formyl core peptide (RT = 4.32 min, MH+=698,2936) and the desired compound (RT= 25.26 += 936,5596) represented below.

MS spectra of the desired compound is presented on Figure 9

The molecular formula of said compound is C45H78N9O10S, and its theoretical mass is 936,55929.

Example 4

Using bioinformatic tools, the inventors identified several additional microorganisms producing antimicrobial compounds of the invention. These microorganisms are listed in Table 10 below. For each of these microorganisms, the sequence of the antimicrobial compound precursor is specified.

Table 10: List of microorganisms producing an antimicrobial compound of the invention

Sequence of the antimicrobial

Accession number, identifier or

Strain compound precursor

reference

(the core peptide is underlined)

Microbacterium arborescens NCBI BioSample: MTLEQLEALDASSEAAEMAAS ND21 SAMN05211039 LGSOSC fSEO ID NO: 23) Uniprot taxon identifier : MSLEQLEALDASSEAAEMAAS

Microbacterium sp. TS-1

1344956 LGSOSC (SEO ID NO: 5)

Oulmi et al. J Bacteriol. 2012 Oct; MID VTDINSLQ AIESHS ATSELL

Tsukamurella sp. 1534

194(19): 5482-5483 ASVSSSGC (SEO ID NO: 18)

MDLTNVIDLQGTEIVADGVELP

Streptomyces aureus DSM 41785

ASGSSEGC (SEO ID NO: 22)

Streptomyces MDLTNVIDLQGTEIVADGVELP

DSM 40541

flavochromogenes ASGSSEGC (SEO ID NO: 22)

MDIADVMDLQGEEVVADGVE

Nocardiopsis chromato genes DSM 44844

LPASTASDGC (SEQ ID NO: 19)

MDLANVMDLQGTEIVADGIEL

Nonomuraea Candida DSM 45086

PASASSETC (SEO ID NO: 21)

MDLTNVMELQGTEIVADGVEL

Streptomyces natalensis

DSM 40357 PASTSSDGC (SEO ID NO: 20)

Claims

1. A bicyclic peptide of formula (I)

wherein

Xi, X₂, X₃, X₄ and X₅ are independently selected and each represents an amino acid, and

2. The peptide of claim 1 , wherein B is hydrogen.

3. The peptide of claim 1, wherein B is a peptide chain comprising, or consisting of, the sequence L-(X)io-E (SEQ ID NO: 24) wherein X represents an amino acid independently selected at each occurrence.

4. The peptide of claim 1 or 3, wherein B is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MS LEQLE ALD AS SE A AEM A (SEQ ID NO: 1) MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2), MID VTNIADLHDID ATS G A AELV (SEQ ID NO: 3), MIDVTDINSLQAIESHSATSELL (SEQ ID NO: 12),

MDIADVMDLQGEEVVADGVELP (SEQ ID NO: 13), MDLTNVMELQGTEIVADGVELP (SEQ ID NO: 14),

MDLANVMDLQGTEIVADGIELP (SEQ ID NO: 15),

MDLTNVIDLQGTEIVADGVELP (SEQ ID NO: 16) and MTLEQLEALDASSEAAEMA (SEQ ID NO: 17).

5. The peptide of claim 4, wherein B is a peptide chain comprising, or consisting of, a sequence selected from the group consisting of MSLEQLEALDASSEAAEMA (SEQ ID NO: 1), MIDVTNIAELHELDSTSASAELV (SEQ ID NO: 2) and MID VTNI ADLHDID ATS GA AEL V (SEQ ID NO: 3).

6. The peptide of any of claims 1 to 5, wherein:

a) Xi is an amino acid selected from the group consisting of A, G, Q, L,W, S and T, preferably A or G, more preferably A; and/or

d) X₄ is an amino acid selected from the group consisting of I, Q, S, N, E, D, W,

H, P and T, preferably Q or N, more preferably N; and/or

e) X5 is an amino acid selected from the group consisting of G, A, S, T, N, R, H, P and D, preferably from G, A, S and T, more preferably G or S, even more preferably G.

7. The peptide of anyone of claims 1 to 6, wherein

X2 is an amino acid selected from the group consisting of L, V, I, G, A, R, T and S, preferably from the group consisting of L, V, I, G, A and T, more preferably from the group consisting of L, V, I, G and A, and even more preferably from the group consisting of L or I and/or X3 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G, A and S, and more preferably from the group consisting of G and S, and/or

8. The peptide of anyone of claims 1 to 7, wherein

X2 is an amino acid selected from the group consisting of L, V, I, G and A, preferably from the group consisting of L or I, and/or

X3 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G and S, and/or

X4 is an amino acid selected from the group consisting of Q and N, and/or

X5 is an amino acid selected from the group consisting of G, A, S and T, preferably from the group consisting of G and S.

9. The peptide of any of claims 1 to 8, wherein

A,

X2 is an amino acid selected from the group consisting of V, I, G, T and A,

X3 is an amino acid selected from the group consisting of A and S,

10. The peptide of anyone of claims 1 to 8, wherein Xi is A, X2 is L and, X3 is G.

11. The peptide of anyone of claims 1 to 8, wherein Xi is A, X3 is G and X4 is Q.

12. The peptide of anyone of claims 1 to 8, wherein Xi is A, X2 is L, X3 is G, X₄ is Q and X5 is S.

13. The peptide of anyone of claims 1 to 9, wherein Xi is A, X2 is I, X3 is S, X₄ is N and Xs is G.

14. The peptide of any of claims 1 to 9, wherein Xi is A, X2 is V, X3 is S, X₄ is S and X5 is G.

15. The peptide of any of claims 1 to 9, wherein Xi is A, X2 is T, X3 is A, X₄ is D and X5 is G.

16. The peptide of any of claims 1 to 9, wherein Xi is A, X2 is T, X3 is S, X₄ is D and X5 is G.

17. The peptide of any of claims 1 to 9, wherein Xi is A, X2 is A, X3 is S, X₄ is E and X5 is T.

18. The peptide of any of claims 1 to 9, wherein Xi is A, X2 is G, X3 is S, X₄ is E and X₅ is G.

19. A method of producing a bicyclic peptide of anyone of claims 1 to 18, comprising:

a) providing a compound of formula (V)

-X, HN

HN (V)

wherein Xi, X₂, X₃, X₄ and X5 are as defined in anyone of claims 1 to 18, and Z a lipophilic moiety, and

b) cleaving Z, thereby producing a bicyclic peptide of formula (I),

and optionally recovering the bicyclic peptide.

20. The method of claim 19, wherein the compound in step a) is obtained from a microorganism producing said compound.

21. The method of claim 20, wherein the microorganism naturally produces said compound or is genetically modified to produce said compound.

22. The method of anyone of claims 19 to 21, wherein step b) is enzymatically performed, preferably using a deacylase, more preferably an aculeacin-A deacylase produced by Actinoplanes utahensis NRRL 12052.

23. A method of producing a bicyclic peptide of anyone of claims 1 to 18, comprising:

providing a linear peptide comprising, or consisting of, the sequence B-X1-S-X2- X3-S-X4-X5-C (SEQ ID NO: 4), wherein B is a peptide chain of a size comprised between 1 and 30 amino acid residues, and Xi, X₂, X₃, X₄ and X5 are as defined in anyone of claims 1 to 18, and contacting said linear peptide with an enzyme or enzymatic extract derived from a microorganism producing a bicyclic peptide.

24. The method of claim 23, wherein B is a peptide chain as defined in any of claims 3 to 5.

25. A peptide comprising, or consisting of, the sequence B-X1-S-X2-X3-S-X4-X5- C (SEQ ID NO: 4), wherein B is a hydrogen, a peptide chain of a size of between 1 and 30 amino acid residues, or an a-amino-protecting group; and Xi, X₂, X3, X₄ and X5 are independently selected and each represents an amino acid.

26. The peptide of claim 25, wherein B is a peptide chain as defined in any of claims 3 to 5 and Xi, X₂, X3, X₄ and X5 are as defined in anyone of claims 1 to 18.

27. A method of producing a lipolanthipeptide, preferably an antimicrobial lipolanthipeptide, comprising:

providing a bicyclic peptide of any of claims 1 to 18, and

adding a lipophilic moiety to said peptide.

28. The method of claim 27, wherein the lipophilic moiety is linked to B, optionally in replacement of B.

29. The method of claim 27 or 28, wherein the lipophilic moiety is R4-W-L- wherein

L is a bifunctional linker, preferably selected from the group consisting of -C(=0)-

, -S02-, -CS-, -0-CS-, -NH-CS-, -PO-, -OPO-, -OC(=0)- and -NHCO-,

R4 is selected from the group consisting of hydrogen and a basic group.

30. A microorganism genetically modified to express a heterologous gene encoding a linear peptide comprising, or consisting of, the sequence B-X1-S-X2-X3-S-X4- X5-C (SEQ ID NO: 4), wherein Xi, X₂, X3, X₄ and X5 are independently selected and each represents an amino acid, preferably as defined in any of claims 6 to 18, and B is selected from the group consisting of hydrogen and a peptide chain of a size comprised between 1 and 30 amino acid residues.

31. The microorganism of claim 30, wherein B is a peptide chain as defined in any of claims 3 to 5.